Role of Ca2 + in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Abstract

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C2C12 murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca2+i, which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) also induced a rise in Ca2+i, but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca2+i induced by PIF and AngII was completely attenuated by the Zn2+ chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn2+. The Ca2+i rise induced by PIF was independent of the presence of extracellular Ca2+, and attenuated by the Ca2+ pump inhibitor thapsigargin, suggesting that the Ca2+i rise was due to release from intracellular stores. This rise in Ca2+i induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca2+i, which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.